The present invention relates to a diagnostic sensor for the detection of influenza virus and to a method of detecting influenza virus with such a diagnostic sensor. This invention was made with government support under Contract No. W-7405-ENG-36 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
The early diagnosis of influenza infection is important for several reasons. One reason is that it is critical to be able to rapidly screen influenza from other infectious diseases in the event of a bio-agent attack. Most scenarios for bio-agent attacks show a slowed response to the recognition that an attack has taken place primarily because diseases such as anthrax and smallpox present flu-like symptoms. Medical personnel do not have a rapid and simple screen for influenza infection and, consequently, victims can be miss-diagnosed as having the flu and sent home. A delay of even a few days in the recognition of a bio-agent attack can have adverse affect on the minimization of the impact of an attack.
Another reason for a rapid diagnostic for influenza is important is in helping to avert a worldwide pandemic in the event that a new strain like the 1918 swine flu appears. Rapid screening with inexpensive fieldable sensors is essential to rapidly pinpoint a new potential outbreak. Although it is also important to specify the strain of the influenza infection, it is first critical to rapidly identify an outbreak and this can only be done using a flexible, inexpensive, fieldable sensor.
Recently, a number of high binding affinity neuraminidase (also known as sialidase) inhibitors have been developed and shown to be quite effective in curing the flu but only if such inhibitors are administered early on in the infection (generally within the first 24 to 48 hours). At resent, these drugs can not be effectively used as there is not a simple diagnostic tool that can be used to detect the influenza virus early enough to effectively use neuraminidase inhibitors. The only technologies currently capable of early diagnosis of influenza are lab-based approaches like ELISA, which are instrument and personnel intensive, expensive, and slow. What is needed is a simple inexpensive diagnosis that can be easily used in either a clinical or field setting and yet have at least the same specificity and sensitivity as ELISA. Accordingly, it is highly desirable to develop a rapid diagnosis for influenza to facilitate the treatment of influenza using such neuraminidase inhibitors.
An optical biosensor system has recently been developed to rapidly detect protein toxins, e.g., cholera, shiga and ricin (see, U.S. Pat. No. 6,297,059, by Song et al., filed Jun. 22, 1999). The integrated optical biosensor developed for the detection of protein toxins was based on proximity-based fluorescence changes that are triggered by protein-receptor binding. In demonstrations of this approach for the detection of cholera and avidin using flow cytometry, it was shown that this technique was as sensitive as ELISA. In contrast to ELISA, such an optical biosensor can be much faster (minutes), simpler (a single step with no added reagents) and robust owing to the stability of the recognition molecules (glycolipids and biotin) and membranes. More recently, an optical biosensor system has been incorporated into planar optical waveguides (see, U.S. patent application Ser. No. 09/598,882, by Kelly et al., filed Jun. 21, 2000) and shown to have sensitivity equivalent to that of flow cytometry. The demonstration of such an optical biosensor using planar optical waveguides provides a path towards the development of miniaturized sensor arrays.
One object of the present invention is adaptation of such a biosensor to sensing applications directed to the detection of influenza virus.
Another object of the present invention is the selection and chemical modification of receptors that bind neuraminidase and that allow attachment of such receptors to membranes together with the incorporation of fluorophores.
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention provides for the detection of tetrameric neuraminidase within a sample, where a positive detection indicates the presence of a target virus within said sample, said sensor including a surface, recognition molecules situated movably at said surface, said recognition molecules capable of binding with said tetrameric multivalent neuraminidase, said recognition molecules further characterized as including a fluorescence label thereon, and, a means for measuring a change in fluorescent properties in response to binding between multiple recognition molecules and said tetrameric neuraminidase.
The present invention further provides a method of method of detecting tetrameric neuraminidase within a sample, where a positive detection indicates the presence of a target virus within said sample, said method including contacting a sample with a sensor including a surface, recognition molecules situated movably upon said surface, said recognition molecules capable of binding with said tetrameric multivalent neuraminidase wherein said recognition molecules include a fluorescence label thereon, and measuring a change in fluorescent properties in response to binding between multiple recognition molecules and said tetrameric neuraminidase.
The present invention further provides for the detection of tetrameric neuraminidase within a sample, where a positive detection indicates the presence of a target virus within said sample, said sensor including a surface, at least two different recognition molecules situated movably upon said surface, said recognition molecules capable of binding with said tetrameric multivalent neuraminidase wherein at least one recognition molecule includes a fluorescence donor label thereon and at least one recognition molecule includes a fluorescence acceptor label thereon, and, a means for measuring a change in fluorescent properties in response to binding between at least two different multiple recognition molecules and said tetrameric neuraminidase.
The present invention further provides a method of method of detecting tetrameric neuraminidase within a sample, where a positive detection indicates the presence of a target virus within said sample, said method including contacting a sample with a sensor including a surface, at least two different recognition molecules situated movably upon said surface, said recognition molecules capable of binding with said tetrameric multivalent neuraminidase wherein at least one recognition molecule includes a fluorescence donor label thereon and at least one recognition molecule includes a fluorescence acceptor label thereon, and measuring a change in fluorescent properties in response to binding between multiple recognition molecules and said tetrameric neuraminidase.
The present invention still further provides a trifunctional composition of matter including a trifunctional linker moiety including as groups bonded thereto (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, said recognition moiety capable of binding with tetrameric multivalent neuraminidase.